Stat3 is a member of the signal transducers and activators of transcription (STAT) protein family that has been shown to be essential for embryonic development and to be activated within cells by a variety of soluble mediators and where it contributes to multiple cell fate decisions including proliferation and differentiation. Using both antisense and dominant negative strategies, Stat3 was shown to be required for TGF-alpha/EGFR- mediated autocrine proliferation of squamous cell carcinoma of the head and neck (SCCHN) cells as well as G-CSF-induced differentiation of murine myeloid cells. Stat3 is recruited to receptor complexes through Stat3 SH2 binding to phosphotyrosine motifs including the consensus motif YXXQ identified within many receptors including the EGFR (at Y1068 and Y1086) and the G-CSFR (at Y704) and to the phosphotyrosine motif YXXC identified thus far only within the G-CSFR (at Y744). Comparison of the Stat3 SH2 domain with non-STAT SH2 domains of known structure revealed the greatest overall homology with v-Src SH2 domain. The pocket within the v-Src SH2 that binds the +3 residue within phosphotyrosine ligands is hydrophobic and preferentially binds phosphotyrosine ligands containing hydrophobic residues at the +3 site. In contrast, modeling of this pocket within the Stat3 SH2 using the recently reported structure of Statl as a template reveals polar residues at the critical sites interacting with the +3 residue of the phosphotyrosine ligand which is consistent with the observations that the preferred phosphotyrosine ligands for Stat3 SH2 are YXXQ and YXXC which feature polar amino acid residues (Q and C) at the +3 position. The validity of this model will be tested in vitro and in vivo in order to understand in greater molecular detail the basis for the Stat3 SH2-phosphotyrosine interaction. The long term goal of these studies is to develop peptidomimetics capable of altering Stat3 interaction with the EGFR and G-CSFR and thereby inhibit SCCHN proliferation and promoting acute myeloid cell differentiation. Towards this end, the Specific Aims of this proposal are: I. To examine the molecular determinants for Stat3 SH2 binding to the phosphotyrosine ligands of the G-CSF and EGF receptors. II. To determine the contribution of the Stat3 SH2-phosphotyrosine binding to G-CSF-induced myeloid differentiation and TGF-alpha/EGFR-mediated SCCHN proliferation.